The benefits of soy protein are well documented. Cholesterol is a major concern with consumers throughout the industrialized world. It is well known that vegetable products contain no cholesterol. For decades, nutritional studies have indicated that the inclusion of soy protein in the diet actually reduces serum cholesterol levels in humans. The higher the cholesterol, the more effective soy proteins are at lowering that level.
Soybeans have the highest protein content of all cereals and legumes. In particular, soybeans have about 40 wt. % protein, while other legumes have 20-30 wt. %, and cereals have about 8-15 wt. % protein. Soybeans also contain about 20 wt. % oil with the remaining dry matter being mostly carbohydrates (35 wt. %). On a wet basis (as is), soybeans contain about 35 wt. % protein, 17 wt. % oil, 31 wt. % carbohydrates, and 4.4 wt. % ash.
In the soybean, both storage protein and lipid bodies are contained in the usable meat of the soybean (called the cotyledon). The complex carbohydrate (or dietary fiber) is also contained in the cell walls of the cotyledon. The outer layer of cells (called the seed coat) makes up about 8 wt. % of the soybean's total weight. The raw, dehulled soybean is, depending on the variety, approximately 18 wt. % oil, 15 wt. % soluble carbohydrates, 15 wt. % insoluble carbohydrates, 14 wt. % moisture and ash, and 38 wt. % protein.
In processing, soybeans are carefully selected for color and size. The soybeans are then cleaned, conditioned (to make removal of the hull easier) and cracked, dehulled and rolled into flakes. The flakes are subjected to a solvent bath that removes the oil. The solvent is removed and the flakes are dried, creating the defatted soy flakes that are the basis of most of the soy protein products. Despite the large number of products on the market, there are only three types of soy protein: flours, isolates, and concentrates.
Soy flours are the simplest forms of soy protein, having a protein content of approximately 50 wt. %. Simply grinding and screening the defatted flakes produces soy flours. This simple processing leaves the soy flour with many of the soybean's characteristics. The lack of processing also makes soy flours highly variable in terms of quality.
Soy flours and grits are still widely produced and are used most often in baked goods, snack foods and pet foods applications where the high flavor profile does not pose a problem. Textured soy flours were an early attempt at simulating or enhancing the texture of meat products. Texturizing does not change the composition of soy flours and reduces the flavor profile only slightly. Their primary applications are inexpensive meat products or pet foods.
Soy concentrates have at least 60 wt. % protein and typically have about 70 wt. % protein. A myriad of applications has been developed for soy concentrates and texturized concentrates in processed foods, meat, poultry, fish, cereal and dairy systems.
Soy protein concentrates are made by removing soluble carbohydrate material from defatted soy meal. Aqueous alcohol extraction (60-80% ethanol) or acid leaching (isoelectric pH 4.5) are the most common means for carbohydrate removal. In both aqueous alcohol extraction and acid leaching, however, essentially all of the protein is rendered insoluble. Protein solubility may be recovered in acid leach products by neutralization.
Isolates are produced through standard chemical isolation, drawing the protein out of the defatted flake through solubilization (alkali extraction at pH 7-10) and separation followed by isoelectric precipitation. As a result, isolates are 90 wt. % protein on a moisture-free basis. Isolates can be made with a high percentage of soluble protein and a low flavor profile. They contain no dietary fiber and are sometimes high in sodium, properties that can limit their application. Their major applications have been in dairy substitution, as in infant formulas and milk replacers.
Bowman-Birk Inhibitor Concentrate (BBIC) has been shown to exhibit inhibitory activity against the malignant transformation of cells under certain conditions and its administration has been shown to affect various forms of cancer.
It has been shown that the enzyme-inhibitor described by Bowman (Proc. Soc. Expd. med, 63:547 (1946)) and Birk et al. (Bull. Res. Council Israel, Sec. A 11:48 (1962) and Biochim. Biophys Acta, 67:326 (1963)), and subsequently referred to as the Bowman-Birk Inhibitor (BBI), can prevent, or greatly reduce, radiologically or chemically induced malignant transformation of cells in culture and in experimental animals.
Yavelow et al. (Proc. Natl. Acad. Sci, USA 82:5395-5399 (1985)) reported that a crude soybean extract, if defatted with acetone, effectively blocked cell transformation in vitro. An active component of this crude extract is BBI. These observations, with epidemiological data, suggested BBI as a putative dietary anticarcinogen, particularly with respect to colon cancer.
Weed et al. (Carcinogenesis, 6:1239-1241 (1985)) discloses that an extract of soybeans containing the Bowman-Birk protease inhibitor added to the diet of dimethylhydrazine (DMH)-treated mice resulted in a significant suppression of odenomatous tumors of the colonic mucosa. DMH-induced colon cancer in mice is generally regarded as an excellent animal model for the human disease, with carcinogen treatment inducing adenocarcinomas of the colon and rectum which are similar to the tumors arising in the human colon suggesting the possibility that a dietary additive of the sort studied might confer some protection against the development of human colon cancer without undesirable side effects. BBI extract and methods for its preparation were as described by Yavelow et al. Cancer Res., 43:2454-2459 (1983); Proc. Natl. Acad. Sci., USA 82:5395-5399 (1985) and Hwang et al. Biochim. Biophys. Acta, 495:369-382 (1977).
Messadi et al. (JNCL 76:447-452 (1986)) demonstrated that a soybean extract containing the protease inhibitor BBI suppresses 7,12-dimethyl-benz[a]anthracene (DMBA)-induced carcinogenesis in the hamster cheek pouch. This oral cancer model, with the use of the hamster cheek pouch carcinogenesis system, has the same histopathology, growth pattern, and precancerous lesions as the most common form of human oral cancer, squamous cell carcinoma. It was shown in this study that hamster cheek pouch carcinogenesis can be inhibited by BBI and suggested that human oral carcinogenesis might respond to BBI in a comparable manner. The BBI preparation used in this study was a crude extract of the inhibitor prepared as described by Yavelow et al. (Proc. Nad. Acad. Sci., USA 82:5395-5399 (1985)).
Baturay et al. (Cell Biology and Toxicology, 2:21-32 (1986)) discloses that a BBI preparation, wherein a crude soybean extract is defatted with acetone, suppresses radiation and chemically induced transformation in vitro, with or without enhancement by the co-carcinogen, pyrene. Yavelow et al., 1985, supra, show that either pure BBI or the BBI extract prepared in accordance with their methods suppresses radiation induced transformation in C3H10TI12 cells. Kennedy et al, Proc. Nat'l. Acad. Sci. USA 1984, 81, 1827-39 reports that either pure BBI or the BBI extract prepared in accordance with their method reduce the levels of chromosome abnormalities in cells of patients with Bloom's syndrome (a genetic disease in which the high levels of chromosome abnormalities are thought to predispose the patients to a higher than normal cancer incidence). Still, other studies suggest that soybean-derived protease inhibitors can have suppressive effects on skin, breast and liver carcinogenesis in vivo.
Kennedy et al. in Anticarcinooenesis and Radiation Protection, edited by Cerutti et al., Plenum Pub. Co., pp. 285-295 (1987), disclosed that BBI suppresses carcinogenesis in various systems using a crude BBI extract prepared by defatting soybeans with acetone. Their results suggested that very low concentrations of BBI-type protease inhibitor preparations would be effective as chemopreventative agents for colon cancer. There was no evidence to suggest that the use of protease inhibitors as chemopreventative agents would be complicated by possible toxicity problems.
St. Clair et al. (Cancer Res., 50:580-586 (1990)) report that the addition of 0.5% or 0.1% semi-purified BBI or 0.1% or 0.01% purified BBI to the diet of DMH-treated mice resulted in a statistically significant suppression of angiosarcomas and nodular hyperplasia of the liver and colon carcinogenesis. The results of this study also indicate that BBI, included as 0.5% of the diet or less had no adverse effect upon the health of the mice but had the capacity to suppress liver and colon carcinogenesis.
Perlmann et al. (Methods in Enzymology, 19: 860-861 (1970)) describes an elaborate method for obtaining the BBI from a defatted soybean extract.
U.S. Patent No. 4,793,996 to Kennedy et al. discloses a process of treating soybeans with acetone, followed by ethanol extraction and acetone precipitation for obtaining BBI. The soybeans may be defatted prior to acetone treatment. In addition, BBI may be further purified by conventional techniques. Kennedy et al. discovered that by treating the soybeans with acetone prior to the ethanol extraction step taught by Perlmann et al., the resulting BBI was more effective in inhibiting the malignant transformation of cells.
U.S. Pat. No. 4,793,996 to Kennedy et al. teaches a process for preparing a crude soybean extract containing a BBI inhibitor of malignant cell transformation which involves defatting soybeans and extracting the inhibitor from the defatted soybeans, and, as an improvement that greatly increases the effectiveness of the BBI inhibitor, defatting the soybeans by bringing them into contact with at least an equal weight of acetone. This process thus produces a crude inhibitor extract which, due to the contact with acetone, nevertheless demonstrates greatly increased effectiveness.
U.S. Pat. No. 5,217,717 to Kennedy et al. teaches ultrafiltration of soy solubles, including a whey protein, to make a BBIC. The ultrafiltration process may be performed alone, or in combination with acetone precipitation, prior to or after the ultrafiltration.
U.S. Pat. No. 5,217,717 to Kennedy et al. also teaches performing two acetone extractions of soy solubles to produce a BBIC, without ultrafiltration. The patentees discovered that spray-drying has no effect on BBI recovery, as measured by chymotrypsin inhibition (CI), used as an indicator for the presence of BBI.
Lunasin is a major component of the Bowman-Birk protease inhibitor from soybeans. Research conducted at the University of California at Berkeley found that lunasin binds to a protein that itself binds to DNA, blocking a step that normally leads to multiplication of cancer cells. Injecting the lunasin-bound protein into cells stops cell division in both normal and cancerous cells. This discovery has lead to the successful use of lunasin in treating human breast cancer cells, and skin cancer in mice, and has spurred research directed to finding delivery systems for lunasin for cancer prevention and treatment.
The prior art has not described a high protein concentrate having high levels of BBI that is obtained from a soy protein source, without acid or alcohol extraction, or acetone precipitation. The prior art also has not described a high protein concentrate having high levels of BBI that is obtained from a fiber-removed soy protein source. The prior art also has not described a high protein concentrate that includes acetone-free BBI. In the present invention, a high protein concentrate having high levels of BBI is produced from a soy protein source, without acid or alcohol extraction, or acetone precipitation.